Curcumin as adjuvant therapy to improve remission in myeloma patients: A pilot randomized clinical trial

Background: The treatment for ineligible transplant multiple myeloma is melphalan prednisone. Curcumin has an anti-inflammatory and antiangiogenesis in cancer-directed to nuclear factor-kappa B (NF-kB) pathway. Interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), and lactate dehydrogenase (LDH) were also involved in the pathogenesis of myeloma. No clinical study has evaluated the efficacy of curcumin in myeloma patients. To evaluate the efficacy of curcumin as adjuvant into melphalan prednisone in myeloma patients Methods: 33 myeloma patients at Dr. Kariadi General Hospital, Semarang, Indonesia during 2016-2017 were randomly assigned single-blindedly into MPC (n=17) and control group (n=16). The MPC group was treated with melphalan 4 mg/m2, prednisone 40 mg/m2 for 7 days, and curcumin 8 gram daily for 28 days. The MP control group was treated with melphalan, prednisone, and placebo. The primary endpoint was the overall remission. Pre- and post-treatment was examined for NF-κB, VEGF, TNF-α, IL-6, LDH, and CRP levels All data analyses were per protocol. Results: There was a significant difference in overall remission between the MPC and MP control groups [75%vs 33.3%, x2=6.89, P=0.009]. A significant decrease of NF-κB, VEGF, TNF-α levels were shown in the MPC group compared with the MP control group. There was a significant decrease in IL-6 levels in a subgroup analysis of the MPC group. TNF-α levels had a significant correlation with remission [OR=1.35; (95%CI=1.03-1.76); P=0.03]. Conclusion: Curcumin has an efficacy in improving overall remission and decreasing NF-κB, VEGF, TNF-α, and IL-6 levels in myeloma patients.

This interesting result make many researchers develop the studies of curcumin as a chemotherapeutic agent. Curcumin has less toxicity and its safety has been approved by the FDA (12). The clinical study of efficacy curcumin has proven to inhibit the progression of monoclonal gammopathy of undetermined significance (MGUS) as well as smoldering multiple myeloma (SMM) (13)(14)(15). Curcumin has an antiinflammatory and anti-angiogenesis in cancer-directed to the target of the NF-κB pathway (16,17,18). The other parameters in disease activity and cytokine involved in the pathogenesis of myeloma were IL-6, VEGF, TNF-α, CRP, and LDH (19,20). The primary outcome of this study was to prove the efficacy of curcumin in the improvement of the remission in myeloma patients. The secondary outcome was to evaluate the effect of curcumin on transcription factor and cytokine levels, including NF-κB, IL-6, VEGF, TNF-α, CRP, and LDH.

Methods
The study was organized at Dr. Kariadi Hospital, Semarang, Indonesia during 2016-2017. Inclusion criteria were as follows: new MM patients, aged over 18 years old, and ineligible transplant. The exclusion criteria were sepsis, severe infection, pregnancy, patients with severe disease (such as acute hepatitis, chronic hepatitis, cirrhosis) or elevation of aspartate aminotransferase (AST) >3 times upper limit normal (ULN), subjects who participated in another study, and poor performance status. The sample size calculation was 32 participants, a total of 16 patients in each group. The α and β errors chosen for these calculations were 0.05 and 0.2, respectively. The power was 80%. Patients with clinically suspected myeloma were evaluated for full blood count, albumin, globulin, urea, creatinine, calcium, protein electrophoresis, immunotyping, bone survey, bone marrow aspiration, and -2 microglobulin. The staging was classified according to the International Staging System (ISS). The diagnosis of myeloma was established according to the IMWG 2014 (21,22).
For the randomization, the participants were randomized into the group with a sealed envelope. A total of 33 patients were allocated randomly in two parallel study groups ( Figure  1). The MPC group (17 patients) was treated with MP regimen (melphalan 4mg/m2, prednisone 40mg/2, for 7 days) and curcumin 8 gram /daily for 28 days. The control group (16 patients) was treated with MP regimen and placebo. Oral melphalan, prednisone was taken as a single dose. Curcumin was taken orally -4 caplets, twice daily. Curcumin 1000 mg (BCM 95, Biocurcem ®) is composed of curcuminoid complex 95% and essential oils from turmeric (23) purchased from PT. SOHO Global Health, Indonesia. Each patient was followed up every 28 days, for a total of 4 cycles. The checklist was used for data collection and was filled in each visit separately for every subject. The contents of the checklist were the patients' profiles (age, sex, education level), and laboratory data, including full blood count (FBC), urea, creatinine, NF-B, IL-6, CRP, LDH, VEGF, and patient group (treatment or control). The physical examination of the patients was performed by a physician in every visit (single blindness). Remission status, NF-B, IL-6, CRP, LDH, VEGF, and TNF- were evaluated after the end of the study. The blood was taken during office hours from the vein and collected in a tube that contains 10 ml EDTA. The blood was centrifuged, then the plasma was collected and kept under -80 • C Refrigerator at GAKI Laboratory, UNDIP Semarang. NF-κB, IL-6, VEGF, and TNF-α levels were measured by ELISA following the manufacturer´s instruction in GAKI Laboratory, UNDIP. CRP and LDH levels were measured at Dr. Kariadi Hospital. Protein M serum and FLC were measured in the Cito laboratory and Prodia laboratory, respectively. Complete blood count (CBC) was measured by flow cytometry method (Cell-Dyn Saphire; Abbott Diagnostics Division, Santa Clara, CA, USA). Serum calcium, CRP, and plasma albumin was measured by homogenous enzymatic colorimetric assay (Dade Behring; Siemens, German). Immunofixation electrophoresis is used to identify the clonality (type) of M-proteins measured by agarose gel. NF-κB (p56 /total) levels were measured by Enzyme-Linked Immunosorbent Assay (ELISA) Kit test, Human. VEGF serum levels were measured by the VEGF ELISA test kit (Quantikine®, Human Soluble VEGF R2 Immunoassay, Catalog Number DVRE 200). IL-6 serum levels were measured by the ELISA kit test (Human IL-6 Immunoassay Catalog Number HS600B). TNF-α serum levels were measured by human TNF-α (ELISA) test kits. The remission was evaluated by bone marrow aspiration, the level of M protein or FLC (free light chain) was classified according to EBMT criteria (for common myeloma type) and IMWG criteria (for light chain myeloma) (24)(25)(26).
BCM-95 ® CG (Biocurcem™) 1000 mg capsules were supplied by Soho Global Health. Biocurcem TM contains Curcuma longa rhizome (Curcuminoid complex 95%) with high bioavailability. Ethical approval and informed consent: This clinical trial has been registered on the ISRCTN registry with study ID ISRCTN14131419. The protocol of the study was approved by the Ethics Committee of the Faculty of Medicine, Diponegoro University, and Dr. Kariadi General Hospital. This study was conducted following the Declaration of Helsinki and all the participants have signed the informed consent. Statistical Analysis: The analysis within groups was performed using the Friedmann test. The analysis of nominal data between groups was done using the chi-square. The analysis of ratio data between groups was done using Mann-Whitney tests or independent t-test. The ratio data was conversed as delta () by comparison with baseline data.
Correlation of variable was done using multiple logistic. The program of SPSS IBM Version 21 was used to analyze the data. The result of the analyzed data was considered significant if p<0.05.

Results
Demography and Characteristics Study: During February 2016 -May 2017, there were 35 myeloma patients in Dr. Kariadi General Hospital, Semarang. Two subjects were excluded from the study. A total number of 33 patients were allocated randomly into two groups. The MPC and MP control groups consist of 17 subjects and 16 subjects, respectively. Four patients in the treatment group died due to sepsis, anemia, and thrombocytopenia in the first-month treatment. Three subjects in the control group died because of sepsis, melena, and anemia in the first-month treatment. One patient from the control group discontinued the study because of flushing after taking medication. One patient in the treatment group was lost of contact in the second month. One patient in the control group died because of anemia and sepsis in the third-month treatment. Table 1 showed the baseline characteristics of the study population. There were no significant differences in the demographics and characteristics of the study population (age, BMI, creatinine, β2m, albumin, calcium, gender, bone lytic lesions, fractures, myeloma type, anemia, the percentage of the plasma cell, stage, and performance status). The consort of the study was shown in Figure 2. Effect curcumin in the remission: The overall remission in the MPC group was better than the control. Table 2 showed the overall remission (OR) of myeloma patients treated with melphalan, prednisone, and curcumin increased to 75.0 %, compared with the MP control group of 20.0%. This result was statistically significantly different with p<0.05 (X 2 = 6.89, df=1, P=0.01). The effect of curcumin in the transcription factor and cytokine levels was shown in table 3. The decrease of NF-κB levels in the MPC group was higher than the MP control group. The table demonstrated the trend of reduction of NF-κB levels that increased in the MPC group. In the 4 th month, the reduction of NF-κB levels reached a significant decrease significantly in the MPC group compared with control (0.66 vs-0.05, P=0.027). The trend reduction of IL-6 increased in the MPC group, and was analyzed with the Friedman test. The reduction of IL-6 level showed a significant decrease in the MPC group (P=0.00), not in the MP   "Eastern Cooperative Oncology Group;" NF-κB, "Nuclear Factor kappa B"; IL-6, "Interleukin-6"; CRP, "C-reactive protein"; LDH, "Lactic Acid Dehydrogenase"; VEGF, "Vascular endothelial Growth Factor"; TNF-α, "Tumor Necrosis Factor-α"   CRP, "C-reactive protein"; LDH, "Lactic Acid Dehydrogenase"; VEGF, "Vascular endothelial Growth Factor"; TNF-α, "Tumor Necrosis Factor-α" Note: multiple logistic regression, VEGF, "Vascular endothelial growth factor"; LDH, "Lactic dehydrogenase"; TNF-α, "Tumor necrosis factor-α"

Discussion
This study proved that there was a difference in overall remission between the MPC and control groups, significantly. The group treated with curcumin has better outcome than the control according to this remission status. Curcumin effects increase overall remission of 75%. Previous studies showed that myeloma patients who took melphalan prednisone had a low overall response rate of less than 50%, with CR below 5% (27,28). To the best of our knowledge, this is the first report that demonstrated that curcumin has an effect in increasing overall remission in myeloma patients.
Numerous studies reported that IL-6 promotes the survival and proliferation of multiple myeloma (MM) cells through the phosphorylation of a cell-signaling protein, STAT3 (19). Interleukin-6 (IL-6) is a cytokine for the defense against acute environmental stress. The pathology in various inflammation and autoimmune disease was caused by persistent dysregulation of IL-6 production diseases (34). Myeloma is a malignancy that is a chronic inflammatory disease (35), it means there is dysregulation of IL-6. This study revealed the decrease of IL-6 level in the group with curcumin. In myeloma patient, the overall survival was significantly different between the low IL-6 and high IL-6 groups, and IL-6 level correlates with the clinical feature and prognosis (36).
The angiogenic activators important in malignancies are vascular endothelial growth factor (VEGF). VEGF has an impact on developing the new vessel in tumors called neovascularization. The new drug was developed against VEGF like bevacizumab approved by US Federal Drug Administration (37,38). Our study showed that curcumin affects decreasing VEGF levels. In this study, we found that curcumin has activities against TNF-α. Previous reports described that curcumin can block the action and production of TNF-α (17). TNF-α is very important in the pathophysiology of human multiple myeloma (39). TNF-α is also highly expressed in tumors and is thought to be proangiogenic cytokine. Paradoxically, at it is in higher doses can be used to destroy tumor vascular (37). TNF-α regulates the tumor environment and it has a pleiotropic effect as a cytokine in the process of apoptosis, angiogenesis, inflammation, and immunity (40).
This study showed, that curcumin does not affect decreasing CRP and LDH levels. C-reactive protein (CRP) was the acute inflammation in interacting with the complement system. These proteins increase in inflammation process and are associated with various diseases including hypertension, atherosclerosis, peripheral vascular disease, diabetes, and metabolic disease (41). The level of serum CRP has an association with the number of osteolytic bone lesions (42). The study reported that curcumin has anti-inflammatory through a significant reduction of IL-6, hs-CRP, and MDA levels (42), not CRP. A high level of serum LDH is a poor prognostic factor in patients with malignancies (43). LDH is a useful marker progression in myeloma (44), but also is found in high levels after physical activities. In Chinese elderly patients with myeloma, LDH has an unfavorable prediction for the outcome (45). Curcumin affects the decreasing NF-κB, IL-6, VEGF, and TNF- levels. Curcumin improves overall remission in myeloma patient. Multivariate analysis proved that TNF- correlates with remission. This study revealed that curcumin improves remission through decreasing TNF- level. Previous studies demonstrated that curcumin affected the transcription and angiogenic factors especially TNF-.
In conclusion the addition of curcumin in myeloma patients treatment with MP regiment will improves the overall remission. Curcumin affects the decrease of NF-κB, IL-6, VEGF, and TNF-α levels. A future study is needed to evaluate the effects of curcumin to the survival and effect of curcumin in combination with another novel agent.